Fluorescent detection of proteins in polyacrylamide gels

ABSTRACT

The mechanism of the UV light-induced reaction between the indole moiety of tryptophan and chloroform, and the structure of the modified tryptophan and polypeptides including such modified tryptophan residues. The excited indole moiety, which is formed upon UV light irradiation, emits a solvated electron which initiates a series of events that yield fluorescent derivatives that have CHO group covalently bound to the indole moiety. These derivatives are herein referred to as formyltryptophan, and are relatively stable. Similar reactions are observed when 5-hydroxytryptophan, 5-fluorotryptophan, or N-methylindolacetate are used in place of tryptophan, or when other haloalkanes, such as trichloracetic acid, trichlorethanol, trichlorethane, bromoform, and iodoactetate are used in place of chloroform. The derivatives can be used in a variety of applications in fluorescence spectroscopy, and for nuclear magnetic resonance, X-ray crystallography, infra-red spectroscopy, circular dicroism and mass spectroscopy. Additionally, the UV light-induced reaction between the indole moiety of tryptophan and haloalkanes can be used to prepare derivatives of tryptophan for chemical cross-linking studies of proteins and peptides.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. ProvisionalPatent Application No. 60/319,099 filed Jan. 25,2002; No. 60/352,225filed Jan. 29, 2002; and No. 60/319,810 filed Dec. 24, 2002; thecontents of each of which are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to polyacrylamide gel electrophoresis andin particular, to a method of visualizing proteins in a polyacrylamidegel.

[0003] Polyacrylamide gel electrophoresis is a well-known technique fordetermining the molecular weight of a protein and for separatingproteins on the basis of their molecular weight. Electrophoresis in theabsence of any denaturing reagent (native-PAGE) results in separation onthe basis of charge and size. It gives an estimation of the size of thefolded protein by reference to proteins of known size. In order todetermine the molecular weight of the polypeptide chain it is necessaryto carry out the electrophoresis in the presence of the anionicdetergent sodium dodecyl sulphate (SDS-PAGE). This detergent not onlycompletely unfolds the protein but interacts with the unfolded chainsuch as to give a constant charge density. This means that separation isonly based upon molecular weight. Calibration of the gel with markerproteins of known molecular weight allows estimation of the molecularweight of unknown proteins.

[0004] Visualization of the proteins separated by SDS-PAGE is typicallycarried out by staining the gel with Coomassie brilliant blue or Amidoblack dyes. Other non-specific visualization techniques include silverprecipitation or staining with fluorescent compounds. Coomassie bluestaining is the most common technique and, similar to other prior arttechniques, typically involves several hours of protein fixation,staining and destaining. There have been many attempts to provideshorter staining protocols.

[0005] The use of fluorescence for protein detection is of course wellestablished in biochemistry. Preelectrophoretic labeling of proteinswith UV-excitable fluorophores, such as FITC (flouresceinisothiocyanate) or bromobimane compounds (1) followed bypostelectrophoretic visualization under UV light has been successfullyused for many years. Also, several methods have been developed forpostelectrophoretic fluorescent labeling with stains such as1-aniline-8-naphthalene sulfonate (2) and o-phthaladehyde (3). All thesemethods involve lengthy labeling steps and each of them has intrinsiclimitations such as altered electrophoretic mobility on native PAGE inthe case of preelectrophoretic labeling or low sensitivity in the caseof postelectrophoretic staining (4). Recently two new fluorescent dyes,SYPRO red and SYPRO orange, have been introduced to detect proteins inSDSPAGE (5). Although quite sensitive, their use is expensive, somewhattime-consuming, and dependent on the presence of SDS.

[0006] There is still a need in the art for further improvements influorescent protein visualization.

SUMMARY OF THE INVENTION

[0007] It is known that a light driven reaction between chloroform andthe indole moiety of tryptophan (Trp) yields products that emit at longwavelengths. This generation of blue fluorescence in biological tissuesamples is used to monitor aging and lipid peroxidation; but the use ofchloroform/methanol in the extraction of tissues and subsequentproduction of long wavelength emitting substances upon irradiation hasbeen noted as a complication in these studies.

[0008] The applicants have recently determined that a UV-light-dependentreaction between Trp in the presence of trichloro compounds such astrichloroacetic acid yields fluorescent products that emit in thevisible spectrum. This reaction is similar to the reaction between Trpand chloroform described in the prior art. This knowledge permitted thedevelopment of a procedure for visualizing proteins after SDS-PAGE whichallowed fluorescent visualization of proteins shortly after completionof electrophoresis and without laborious labeling or staining steps.

[0009] In one aspect, the invention may comprise a method of visualizinga protein in a polyacrylamide gel, comprising the steps of

[0010] :(a)incorporating a haloalkane into the gel, either pre- orpost-electrophoresis, which haloalkane reacts with tryptophan residuesin the proteins to form fluorescent compounds when illuminated withultraviolet light; and

[0011] (b)electrophoretically migrating the protein in the gel; and

[0012] (c)illuminating the gel with ultraviolet light; and

[0013] (d)detecting the fluorescence of the formed compounds.

[0014] Preferably, the haloalkane is a trichloroalkane and morepreferably, the trichloroalkane is selected. from the group consistingof chloroform, trichloroacetic acid and trichloroethanol, or mixturesthereof. In one embodiment, the haloalkane is incorporated into the gelby soaking the gel (post-electrophoresis) in a haloalkane solution. Inanother embodiment, the haloalkane is incorporated into the gelpre-electrophoresis before polymerization of the gel. In anotherembodiment, the haloalkane may be incorporated into the buffer in whichthe proteins are dissolved pre-electrophoresis.

[0015] In another aspect, the invention may comprise a polyacrylamidegel for electrophoretically separating proteins by molecular weight, thegel comprising a haloalkane which reacts with tryptophan residues in theproteins to form compounds which fluoresce when subjected to ultravioletlight.

[0016] In another aspect, the invention may comprise a kit for forming apolyacrylamide gel for electrophoretically separating proteins bymolecular weight, comprising a haloalkane for incorporation into the gelwhich reacts with tryptophan residues in the proteins to form compoundswhich fluoresce when subjected to ultraviolet light.

BRIEF DESCRIPTION OF DRAWINGS

[0017]FIG. 1A shows a recombinant protein (lanes 1 and 5) and three ofits mutants (Mutant 1, lanes 2 and 6; Mutant 2, lanes 3 and 7; Mutant 3,lanes 4 and 8) were resolved on SDSPAGE. Samples in lanes 14 weretreated with β-mercaptoethanol prior to loading on the gel. The proteinsin the gel were visualized by soaking the gel in trichloroacetic acid(TCA) post electrophoresis followed by ultraviolet (UV)transillumination. FIG. 1B shows the same gel stained overnight withCoomassie brilliant blue (CBB) R-250. Monomeric recombinant protein isindicated by a filled arrow. Predominant oligomer is indicated by anopen arrow. FIGS. 1C(TCA) and 1D (CBB) show the results of twoEscherichia coli clones tested for expression of a recombinant protein.Lanes 14, clone 1; lanes 58, clone 2. Lanes 1 and 5, total bacterialprotein prior to induction; lanes 2 and 6; total bacterial protein afterinduction; lanes 3, 4, 7, and 8, affinity-purified recombinant proteinisolated by two slightly varying protocols. Recombinant protein isindicated by a filled arrow. M-molecular weight markers.

[0018]FIG. 2A shows a gel wherein bovine serum albumin (BSA) was loadedon the gel in the following amounts: Lane 1250 μg , lane 2125 μg, lane350 μg, lane 425 μg, lane 55 μg, lane 62.5 μg, lane 70.5 μg. M,molecular weight markers. Protein was visualized by the TCApost-electrophoresis soak method. FIG. 2B shows the same gel stainedovernight with CBB R-250 (B).

[0019]FIG. 3 shows gels visualized under different conditions todetermine optimum sensitivity. Bio-Rad low-molecular-weight standards at1.2, 3.0, and 6.0 μg per lane (which corresponds to 0.2, 0.5, and 1.0 μgper band) were run on thin (0.75 mm thick) 12% polyacrylamide gels.After electrophoresis portions of gels A and B were soaked in 5% TCA,10% TCA, 20% TCA, or 50% methanol:water saturated with chloroform for 10min, washed with water at least five times over a 10-min period, andthen illuminated for 5 min on the transilluminator. Gel C has been CBBstained after treatment with saturated chloroform as described above forgel B. Pictures were taken With a transilluminator digital camera usinga 4-s integration time.

[0020]FIG. 4 shows that protein fluorescence is largely specific for TCAin contrast with other acids. BSA (0.1 μg/lane) was resolved on SDSPAGE,gel was cut in strips, and each strip was soaked in 20% aqueous solutionof an acid as indicated below. Strips were photographed in UV light (A)and then stained overnight with CBB R-250 (B). Lane 1, TCA; lane 2,acetic acid; lane 3, hydrochloric acid; lane 4, sulfuric acid; lane 5,nitric acid. Fluorescence in lanes 25 was not detectable by naked eye.

[0021]FIG. 5 shows fluorescence monitoring of the reaction of Trp in thepresence of TCA. Emission spectra of 10 mM Trp in the presence of 100 mMTCA with 280-nm excitation spectra. Scans were begun at 0, 2, 4, 6, 10,14, and 18 min. The inset shows the excitation spectra with 450-nmemission that was obtained after the scanning.

[0022]FIG. 6 shows fluorescence monitoring of the reaction of Trp andn-acetyl-L-tryptophanamide (NAWA) in the presence of HCl. Emissionspectra of 10 mM Trp plus 100 mM HCl with 280-nm excitation spectra.Scans were begun at 0, 10, and 18 min. The line near zero is for 10 mMNAWA plus 100 mM HCl at the 18-min scan and is also magnified 10-fold(designated 103) for clarity.

[0023]FIG. 7 shows a comparison between trichloroethanol (TCE) addedpre-electrophoresis in gel visualization and Coomassie Brilliant Blue(CBB) staining. FIG. 7A shows the detection of low molecular weightstandards with 0.5% TCE in 12% SDSPAGE. FIG. 7B shows a CBB stain of thegel from FIG. 7A. FIG. 7C shows an SDS-PAGE with 0.5% TCE of 0.25 μg and0.5 μg DmsD:His6 (lane 1 and 2) and TehB:His6 (lane 3 and 4) visualizedwith UV light and then western blotted with antiHis6. A duplicate gelwas stained with CBB. FIG. 7D shows a SDS-PAGE with 0.5% TCE (v:v) of0.25 μg and 0.5 μg EmrE (lane 1 and 2) visualized in UV light and thenstained with CBB.

[0024]FIG. 8 is a graph showing the relationship between TCEconcentration and fluorescent intensity. The data was obtained from aSDS-PAGE gel of low molecular weight standards conducted with variouspercentages (vol/vol) of TCE in the gel before electrophoresis.Intensities per μ g are from the sum of intensities from phosphorylaseb, albumin, ovalbumin and trypsin protein bands. The error bars are thestandard deviations.

[0025]FIG. 9 shows the linear dynamic range of the TCE in gel method andthe effect of tryptophan content. Intensities are from a 12% SDS-PAGEwith 0.5% (vol/vol) TCE in the gel. FIG. 9A shows the sum of intensitiesfrom phosphorylase b, albumin, ovalbumin and trypsin with 0.25, 0.5,1.0, 1.5 μg per band were used. FIG. 9B shows the intensity per μ gobtained from the serum albumin (0.8%), ovalbumin(1.3%), trypsininhibitor (1.8%), phosphorylase b (2.3%), carbonic anhydrase (4.5%),EmrE (5.0%), lysozyme (7.8%), and DmsD (8.7%) using 0.25 and 0.5 μg ofprotein. The error bars are the standard deviations.

DETAILED DESCRIPTION

[0026] The present invention provides for methods of visualizingproteins by means of an ultraviolet (UV) light dependent reactionbetween tryptophan (Trp) and a haloalkane, which results in productwhich fluoresce in the visible range. When describing the presentinvention, all terms not defined herein have their common art-recognizedmeanings.

[0027] The present invention comprises a method to rapidly visualizeproteins in polyacrylamide gels following the fixation step in astandard staining procedure following electrophoresis. The methodutilizes the fluorescence of products of the reaction between modifiedtryptophan residues present in the proteins and trichlorocompounds, suchas chloroform or TCA. Since TCA is commonly used as a general purposefixative in staining protocols, this method effectively does notintroduce any additional steps in the conventional CBB staining routine.In short time a researcher gets a first glimpse at the results of theelectrophoresis run and is able to visualize the most abundant proteinspecies. In many cases, this preview may be sufficient to answer mostquestions regarding the presence, relative abundance, mobility, andaggregation of well-represented proteins. Following the initialvisualization, the gel can be further stained or blotted withtraditional methods, thus avoiding spending extra “hands-on”time on fast“hot stainer and destainer” protocols, working with hot solvents andgenerating unpleasant odors. Generation of fluorescent protein bandsinvolves a photochemical reaction between tryptophan residues withinproteins and haloalkanes.

[0028] As used herein, the term “haloalkane” refers to aliphatichydrocarbons substituted with at least one halogen atom. Thehydrocarbons may include alcohols, acids and amides. Preferably, thehaloalkane is a trihaloalkane including without limitation chloroform,bromoform, iodoform, 1,1,1-trihaloalkanes, 1,1,1-trihaloalkanols such astrichloroethanol, trichloroacetate and tribromoacetate. Haloalkanes mayalso include single and di-substituted haloalkanes such as iodoacetate.As used herein, “ultraviolet light” or “UV light” refers toelectromagnetic radiation beyond the violet end of the visible spectrum.The wavelength of UV light may range from less than about 200 nm toabout 400 nm. As used herein, to “fluoresce” means to emit lightdetectable by the naked eye or by an imaging system such as a film ordigital camera.

[0029] In a basic embodiment, proteins in a polyacrylamide gel may bevisualized by incorporating a haloalkane into the gel, which haloalkanereacts with tryptophan residues in the proteins to form compounds whichfluoresce when illuminated with ultraviolet light. Thereafter, when thegel is illuminated with ultraviolet light, the fluorescent proteins maybe detected by the naked eye or by using imaging devices. The haloalkanemay be incorporated into the gel either pre-electrophoresis orpost-electrophoresis.

[0030] Therefore, in one embodiment, after proteins have beenelectrophoretically resolved in a gel with a SDS-PAGE procedure, the gelmay be soaked in a haloalkane solution and then illuminated with UVlight to produce fluorescent protein bands. In another embodiment, thehaloalkane may be added directly to the gel prior to electrophoresis.Preferably, the haloalkane may be added to the gel prior topolymerization. The applicants have found that this improvement mayallow the speed and sensitivity of the method to be improved so that aslittle as 0.1 μg of typical globular proteins can be visualized veryshortly after completion of electrophoresis, with minimal processing ofthe gel. For proteins having a high percentage of tryptophan, a methodas described herein may detect as little as 0.01 μg of such proteinswhich is much more sensitive than Coomassie brilliant blue (CBB). Themethods described herein may be particularly useful to detect integralmembrane proteins which do not stain well with CBB. A comparison of TCApost-electrophoresis soak and CBB staining may be seen in FIG. 1.

[0031] The haloalkane may be (but is not limited to) trichloroethanol(TCE), trichloroacetic acid (TCA) or chloroform. Preferably, thehaloalkane comprises TCE, alone or in combinations of TCE and TCA. TCEis most preferred as it is less volatile than chloroform and thus lesslikely to be inhaled. Furthermore, TCE is less corrosive than TCA.

[0032] Compared to SYPRO Ruby ™, the most sensitive of the SYPRO ™series (6), the methods herein using TCE are less sensitive; however, itis much faster and far less expensive. Furthermore SYPRO Ruby ™, toutedas the best for 2-dimension electrophoresis, has a similar bindingmechanism as CBB, so it can be expected that proteins not efficientlyvisualized with CBB will not be visualized by SYPRO Ruby ™ either.

[0033] With post-electrophoretic embodiments, the gel may be soaked in ahaloalkane solution varying from about 1% to about 30% haloalkane,preferably between about 5% to about 20% haloalkane, and most preferablyabout 10% haloalkane. In one embodiment, particularly good results maybe obtained using 10% TCE in methanol:water (1:1) in the soakingprocedure. The soaking step may vary from 1 minute to about 10 minutes,and preferably about 5 minutes in length. The variables of haloalkaneconcentration and the length of soaking time are not essential elementsof the invention and may be varied to obtain optimal results in eachparticular case with simple experimentation.

[0034] Incorporating a haloalkane into the gel prior to electrophoresishas been found to provide slightly greater intensity and fastervisualization, as a result, of eliminating the soaking step referred toabove. In comparison CBB methods take at least 30 minutes to stain andseveral hours to destain. A comparison of the results may be seen inFIG. 7A and 7B. The haloalkane may be added to the gel prior topolymerization in a suitable concentration which may vary from about0.02% to about 2.0% (v:v) or more. The applicants have found thatincreasing TCE concentration in the gel tends to increase the bandintensity up to about 0.5% TCE (v:v) after which the intensity no longerincreases, as may be seen in FIG. 8. Again, the concentration of thehaloalkane in the gel is not an essential element of the invention andmay be varied to obtain optimal results with simple experimentation.

[0035] The pre-electrophoresis incorporation of a haloalkane methodoffers a linear dynamic range from 0.2 μg to 2 μg of protein with acorrelation coefficient of 0.98 (see FIG. 9A). The sensitivity limit ofthe method for globular proteins with typical percentages of tryptophan(0.8 to 2.3%) is approximately 0.1 μg . All the proteins in lowmolecular weight standards are detectable at this limit. For carbonicanhydrase, which has a high tryptophan percentage (4.5%), thesensitivity limit is 0.01 μg. Testing the linear dynamic range forspecific proteins with the TCE staining method demonstrates that theupper limit of the linear dynamic range is lower with proteins of highertryptophan percentage. Thus the dynamic range is shifted to lowerprotein amounts, so both the upper limit and the sensitivity limit islower.

[0036] The intensity of protein bands increases as the percentage oftryptophan increases as shown in FIG. 3B. The intensity appears toincrease to a maximum intensity and then level off. Phosphorylase b(2.3% Trp, 97 kDa) does not fit the pattern. A possible explanation forthis is that phosphorylase b band is thin and once all the pixels arelit up in the small area an increase in intensity cannot be measured.Similar studies done using the method in which TCE was soaked into thegel after electrophoresis. resulted in wider phosphorylase b bands andconsequently the intensity of the band fit the pattern better (resultsnot shown). When fitting a linear line to the pattern (intercept set tozero) the correlation coefficient is 0.78, which is statisticallysignificant using a 1 tailed t-test and a 95% confidence limit.

[0037] The haloalkane reaction of tryptophans does not interfere withsubsequent blotting of proteins from the gel using known blottingprocedures. As shown in FIG. 7C, proteins bands which fluoresce under UVillumination may also be detected by a Western blot, subsequent to thefluorescent detection procedure.

[0038] Haloalkane-UV modified tryptophan protein detection has thepotential to be especially beneficial for detection of integral membraneproteins. The membrane spanning regions of integral membrane proteinshave a higher percentage of tryptophans then globular proteins (7,8).

[0039] The modified tryptophan visualization of the present invention isuseful because the speed of the visualization technique allows nearlyimmediate protein detection in PAGE. The methods of the presentinvention ay be implemented in automated and high throughputtechnologies for proteomics. Visualization techniques in accordance withthe present invention may complement other staining techniques to allowdetection of proteins not stained efficiently by these methods.

[0040] Although the invention has been described in relation toSDS-PAGE, the methods described herein are applicable to non-denaturinggel electrophoresis.

EXAMPLES

[0041] The following examples of general methods and materials areintended to illustrate specific embodiments of the invention and not tolimit the invention claimed below.

Example 1

[0042] Post-Electrophoresis Incorporation—Materials and Methods

[0043] Electrophoresis was performed in each case using anelectrophoresis apparatus from Owl Scientific (Portsmouth, N.H.) with 153 20 3 0.15-cm gel cassettes or a Mini Protean II ™ system from Bio-Rad(LaJolla, Calif.). Upon completion of electrophoresis, the gel wasremoved from the cassette and placed in the haloalkane solution. Aftersoaking from 5 to 10 minutes , the haloalkane solution was decanted, andthe gel was rinsed several times with tap water to remove residualsolution and then placed in water to prevent drying. If trichloroaceticacid (TCA) is used, it is important to rinse the gel to remove excessTCA prior to protein visualization since TCA is corrosive and willdamage the UV transilluminator. To visualize proteins, the gel wassubjected to UV illumination using a standard UV box. During the courseof UV irradiation, resolved proteins became visible as bluish-greenbands against the background of pale blue gel matrix. Fluorescencedevelops gradually and the bands become fully visible after 1 to 5 minof UV exposure. Depending on the UV transilluminator used and the geldocumentation system available, differences in the ability to photographthe results will exist. A Bio-Rad Gel Doc ™ system and an UltraLum ™transilluminator (300-nm) or a mounted photographic camera with f5 2,58-mm objective equipped with an orange UV filter proved suitable.Exposure times of 5 to 20 seconds worked best for the photographicimages shown in this manuscript, using ASA 400 TMAX ™ black and whitefilm (Kodak). Illuminating the gel from the side or placing it on top ofa UV box gave equivalent results for the purpose of visualization andphotography. Fluorescence, once developed, is stable for several hoursin room light and is immediately visible upon repetitive UV irradiation.Gels may be irradiated in the transilluminator for as long as 30 minwithout any noticeable loss of fluorescence. It is also possible to soakgels in water after TCA treatment for an extended period of time: wehave allowed up to 15 min between removal of TCA and visualization underUV without any loss of sensitivity. Following visualization under UV,the gel can be stained using standard CBB protocol to visualize lessabundant protein species.

[0044] All excitation and emission spectra were recorded with aFluorolog-3 ™ (ISA Jobin-Yvon Spex) fluorometer using a 450-W Xe lampwith 5-nm slitwidths for both the excitation and the emission, whilestirring in the 1.00-cm cuvette with a small bar at about tworevolutions per second. Spectra were taken in a temperature controlledenvironment at 20° C. Because the light-dependent reaction is driven bythe excitation light, repetitive scans were made at a uniform scan rateat 2-min intervals to follow the reaction.

Example 2

[0045] Comparison of Fluorescent Detection with CBB Staining.

[0046]FIG. 1 shows results which validate the utility of this method. InFIGS. 1A and 1B, approximately 20 μg of a recombinant protein and threeof its mutants, all with electrophoretic mobility corresponding to 46kDa, were loaded on the gel in sample buffer either with or withoutadded β-mercaptoethanol (BME). FIG. 1A is a picture of the gel soaked in10% TCA for 5 minutes and rinsed three times with tap water. FIG. 1B isthe same gel after conventional CBB staining. It is evident thatwild-type (wt), as well as mutants 1 and 2, form aggregates of a highermolecular weight in the absence of BME, while mutant 3 does not. Theseproteins could be visualized by UV illumination 10 min after thecompletion of the PAGE run. In FIGS. 1C (TCA) and 1D (CBB), anexpression of a recombinant protein by two bacterial clones and itspurification were followed. The purifications were run in duplicate. Thegel demonstrates that clone 1 does not express the target protein, whileclone 2 does. The purified fraction contains the major band atapproximately 45 kDa and a minor contaminant or degradation product atapproximately 20 kDa. The bands on the gels were visible in less than 10minutes.

[0047]FIG. 2 demonstrates the sensitivity of this method using thick(1.5 mm) gels. Bovine serum albumin (BSA) was loaded on the gel: Lane1250 μg , lane 2125 μg, lane 350 μg, lane 425 μg, lane 55 μg, lane 62.5μg, lane 70.5 μg. M, molecular weight markers. The gel was run andresolved BSA was visualized using the described TCA protocol. As littleas 2.5 μg of BSA could be detected visually and as little as 0.5 μg ofBSA could be detected photographically. The band in FIG. 2, lane 5 (5 μgBSA), was clearly visible, while the band in FIG. 2, lane 6 (2.5 μgBSA), was somewhat harder to see, although still distinguishable.

[0048] The band in FIG. 2, lane 7 (0.5 μg BSA), was not visible by thenaked eye but was detectable by photography. It should be noted thatblack and white photography is in this case more sensitive than nakedeye. The protein band in A, lane 7, is clearly visible on thephotograph, but was indistinguishable by visual observation.

[0049]FIG. 3 illustrates trials run on thin gels (0.75 mm). Themolecular weight standards used in FIG. 3 are the same standards used inFIG. 2. However, in FIG. 2 as much as 3 μg of protein per band ofmolecular weight markers were used to give the same intensity of bandsas is shown in FIG. 3, whereas in FIG. 3 the maximum load was 1.0 μg perband. As well, three different TCA concentrations were used, as well asa chloroform solution. As may be seen in FIG. 3, we determined that TCAconcentration can be decreased to 5% without loss of sensitivity andthat chloroform can be used in place of TCA. In the window of thetransilluminator, the bands on the gel soaked in chloroform and thenilluminated are somewhat brighter than those treated with TCA, but theoptics of the transilluminator give them about the same magnitude whenphotographed. While we had to use a mounted photographic camera toobtain images of thick (1.5 mm) gels, the protein bands on thin (0.75mm) 12% polyacrylamide were sufficiently intense that they were recordedusing a digital camera attached to the transilluminator. Comparisonbetween FIGS. 2 and 3 suggests that the use of thinner gels, as well asmore sophisticated documentation equipment appears to result in greatersensitivity.

Example 3

[0050] Fluorescence Caused by Reaction with Haloalkane

[0051] The applicants tested different acids to check whether thiseffect is solely pH dependent or if it is mediated by a chemicalreaction with TCA. As shown in FIG. 4, a TCA soak produced brightvisible fluorescence, while exposure of gels to other acids resulted ina dim glow, not visible by the naked eye against the UV background.Black and white photography, however, was able to record this weakfluorescence. The possibility that the observed fluorescence is causedby heat from the transilluminator rather than UV irradiation waseliminated by performed experiments in which fluorescence was developedusing a hand-held UV lamp (data not shown). Also, band patterns could berecorded with a portable UV box positioned to the side of the gel,rather than immediately beneath it. In both cases heat transfer from theUV source to the gel was minimal.

[0052] The applicant further determined that heating of TCA-soaked gelsin the absence of UV exposure does not produce visible band pattern(data not shown). In addition, fluorescence spectra shown in FIGS. 5 and6 were obtained in a temperature-controlled environment at 20° C. Takentogether, these data suggest that the observed fluorescence of separatedproteins is caused by UV illumination and is not induced by heating ofthe gel. The fluorescence observed after TCA or chloroform treatment ofthe gels can be explained in terms of fluorescent properties of productsof a light-driven reaction of tryptophan with the trichlorocompound.

[0053] The fluorescence of tryptophan at 350 nm in aqueous solution isnot visible. However, Vorobei et al. have shown that tryptophanundergoes a light-driven reaction with chloroform to yield products thatemit in the visible part of the spectrum (9, 10). They also used14C-labeled chloroform to show that the chloroform carbon is covalentlyattached to the tryptophan (10) in the products of this reaction. Recent1 H NMR evidence has established unequivocally that the chloroformhydrogen is also covalently attached and implies that the products ofthe reaction contain a CHCl attached to the indole ring (11). FIG. 5shows that tryptophan illuminated with UV light at 280 nm in thepresence of trichloroacetic acid leads to a decrease in the indolefluorescence at 360 nm and the production of an emission band atapproximately 420 nm. Similar experiments in which chloroform andN-acetyl-L-tryptophanamide (NAWA) or Trp are illuminated also show adecrease of the indole fluorescence and increase in long wavelengthemission (23). Although the emission peak of the products of thelight-dependent reaction of TCA and Trp is barely in the visible rangeat 420 nm, the long wavelength side of the band gives sufficient lightto be observed with the naked eye. The environment of theSDS/protein/qel matrix may also give a red shift to make even more ofthis band visible. With chloroform, the emission peak produced is atsomewhat longer wavelengths at about 480 nm, which is easily visible tothe naked eye.

[0054] The excitation spectra in the inset of FIG. 5 for the products ofthe reaction between tryptophan and trichloroacetic acid shows a peak atabout 290 nm with a shoulder at about 320 nm. The UV light produced inthe transilluminator, which is primarily above 300 nm, would effectivelyexcite via the 320-nm shoulder on the long wavelength side of the 290-nmpeak. The fact that the fluorescence observed on the gels appearsgradually over the course of UV irradiation supports the involvement ofthe UV-light-driven reaction between proteins embedded in the gel matrixand TCA. The asymmetry of the long-wavelength emission band indicatesthat multiple products are formed by the lightdriven reaction of Trp inTCA. The lack of an isobestic point near 400 nm also implies that aproduct initially is formed that undergoes further reactions. Althoughthese products probably include the known weakly fluorescent degradationproducts of tryptophan: kynurenine, N-formyl kynurenine, and, withlesser yield, hydroxykynurenine (2428), there are new products formed inthe presence of trichlorocompounds in which the indole ring has beenderivatized that have more intense emission in the visible region(9-11).

[0055] Further confirmation that the species that we are able tovisualize is a tryptophan product can be found in FIGS. 1 and 3. We usedlow-molecular-weight markers from Bio-Rad (LaJolla, Calif.). In FIG. 1the molecular weights of resolved protein markers are 97, 66, 45, 31,and 21 kDa, whereas the 12% gel in FIG. 3 also resolves the 14-kDaprotein marker. As seen on CBB-stained gels (FIGS. 1B and 3C), mass loadof 97-, 66-, and 31-kDa markers is approximately the same. However, the31-kDa marker shows best on the UV-visualized gel (FIGS. 1A, 3A, and3B), with 97 kDa following and 66 kDa marker almost invisible. Thisorder of fluorescence intensity for the protein markers matches thetryptophan contents of the respective proteins. Bovine carbonicanhydrase (31.0 kDa) contains 4.5% Trp by weight, rabbit musclephosphorylase B (97 kDa)-contains 2.3% Trp, while BSA (66 kDa) containsonly 0.8% Trp. The ovalbumin (45 kDa) band is always more diffuse andfainter than the other bands and has only 1.3% Trp. Trypsin inhibitor(22 kDa) with 1.8% Trp has a low mass load but a reasonably intensefluorescent band in FIGS. 3A and 3B. Lysozyme (14 kDa), with 7.8% Trp,has the most intense fluorescence band in FIG. 3 even though it is lessintense on the CBB-stained gel (FIG. 3C).

[0056] We were able to document weak fluorescence of proteins after UVillumination with sulfuric, nitric, hydrochloric, and acetic acids.However, this fluorescence is too weak to be useful for visual proteindetection. If Trp in HCl is illuminated in the fluorometer (FIG. 6) thedecrease in indole fluorescence is much slower than was the case for TCAand no long-wavelength emission is observed. However, when NAWA in HClis illuminated, a very weak broad long-wavelength emission band isobserved (shown at 10-fold magnification in FIG. 6) from a reactionwhich must involve the amide part of the NAWA since it is not observedwith Trp. This is consistent with the results of Holt et al. (14), whoconcluded that the light-dependent degradation of Trp containingpeptides leads to significant amounts of products with higher molecularweight than the reacting peptide due to crosslinking.

Example 3

[0057] Pre-Electrophoretic Incorporation of Trichloroethanol (TCE)

[0058] Low molecular weight standards, 1.5, 3.0, 6.0, and 9.0 μg perlane which corresponds to 0.25, 0.5, 1.0, and 1.5 μg per band, wereseparated on a 12% SDS-PAGE gel Protean II gel system (Bio-RadLaboratories, Hercules, Calif., USA) as per the standard Laemmli method(17). Low-molecular-weight standards were from Bio-Rad Laboratories.They contained phosphorylase b (97 kDa, 2.3% Trp), serum albumin (66kDa, 0.8% Trp), ovalbumin (45 kDa, 1.3% Trp), carbonic anhydrase (31kDa, 4.5% Trp), trypsin inhibitor (21 kDa, 1.8% Trp), and lysozyme (14kDa, 7.85 Trp). All tryptophan percentages are calculated as percentageweight. Other purified protein samples loaded were EmrE, DmsD:His6, andTehB:His6. EmrE was purified according to Winstone et al. (18).DmsD:His6 (19) and TehB:His6 (20) were purified with a nickel agarosecolumn followed by size exclusion chromatography. For CBB method the gelwas then stained in Coomassie brilliant blue dye overnight, followed by5 hours destaining. For the TCE in gel method, 0.5% vol/vol TCE (Sigma)was dissolved in the buffer and then acrylamide, SDS, ammoniumpersulphate and TEMED were added to polymerize the separating gel. Thestacking gel is prepared as usual.

[0059] Proteins were visualized by placing the gel on a UVtransilluminator and irradiating the gel for 2 to 5 minutes, duringwhich time the protein bands become visible as bluish-green bandsagainst a pale blue background of the gel matrix. An UltraLum ™Electronic UV transilluminator (300 nm) with COHU ™ High PerformanceMonochrome CCD Camera (Rose Scientific), was used to take photographs ofthe gel. Pixel intensity in bands was determined using Scion Image V1.62software (ftp: zippy.nimh.nih.gov). The density of the background aboveand below a band is averaged and the density of the band is subtractedfrom this giving the intensity of the band.

[0060] It is apparent that visualization with TCE-ultraviolet lightmodified tryptophan is in some cases more sensitive than CBB and faster.FIGS. 7A and 7B shows 12% SDS-PAGE gels loaded with low molecular weightstandards detected with 0.5% TCE in the gel and with CBB staining.Comparison of panel A with B shows that TCE is more sensitive than CBBstaining. The sensitivity limit of CBB is sensitive in the sub μ g rangeand the limit changes for different proteins (21). The presence of TCEin gels during electrophoresis does not appear to impair the mobility ofthe proteins because adding TCE to the gel before electrophoresis doesnot shift the protein bands (results not shown). All of the proteinbands are clearly visible when only 0.25 μg per band of standards areloaded in a 0.5% (v/v) TCE gel. An alternative method is to soak the gelpost-electrophoresis with 10% TCE. In this case the bands are almost asintense (results not shown) as when TCE was put into the gel, but addingTCE to the gel during polymerization significantly decreases theprocessing time.

[0061] Haloalkane-UV modified tryptophans visualization may beespecially useful for membrane proteins. DmsD (19) (8.7% Trp), aperipheral membrane protein, contains 8.7% percent tryptophans and isvisualized with TCE better than CBB as shown in FIG. 1B. TehB (20), asoluble protein, appears to be visualized with equal intensity by bothmethods. An integral membrane protein, EmrE (18) (5.0% Trp) at 0.5 μg,is not seen when stained with CBB as shown in FIG. 1C. In comparison theTCE in gel technique gives very intense bands at just 0.25 μg as shownin FIG. 1C.

Example 4

[0062] Pre-Electrophoretic Incorporation: Optimization and Sensitivity

[0063] In optimization studies 0.02, 0.05, 0.1, 0.2, 0.5, 1.0 and 2.0%TCE (vol/vol) were added to the gel before polymerization. For the TCEstaining the 12% SDS-PAGE was run and then soaked in 10% TCE (v/v) inwater:methanol (1:1) for ten minutes. Then the gel was washed in waterand visualized as described above.

[0064] Calculating the intensities for FIG. 2 and 3A, the sum of theintensity of phosphorylase b, albumin, ovalbumin and trypsin bands wasused. These were chosen because they contain percentages of tryptophannear the average for soluble proteins. Four sets of 0.25 μg and 0.5 μgbands were used to calculate intensity per μ g.

[0065]FIG. 8 shows that maximum intensity is reached with a TCEconcentration of 0.5% and that lower concentrations have a lowerintensity while higher concentrations do not have higher intensities.

[0066]FIG. 3A shows that intensity increases nearly linearly with theamount of protein up to about 2 μg per band. FIG. 3B shows thatincreasing Trp content in the protein correlates with higher intensitiesper μ g of protein.

Example 3

[0067] Western Blots After Fluorescent Visualization

[0068] In addition to rapid protein detection, the methods describedherein allows for visualization of proteins before western blotting.FIG. 7C shows proteins visualized by TCE in gel followed by a westernblot. This will allow for confirmation that an appropriate proteinpattern is seen before performing a western blot procedure. In additionthis demonstrates that TCE in the gel does not hinder transfer ofproteins to nitrocellulose.

[0069] For the western blot shown in FIG. 7C, immediately aftervisualizing 12% SDS-PAGE with 0.5% TCE, the gel was electroblotted tonitrocellulose (TransBlot). The blot was then blocked overnight with 5%milk in Tris Buffer Saline. The blot was then incubated with the primaryantibody, antiHis6 (Cedarlane Laboratories Ltd, Hornby, ON, CAN), washedand incubated with goat antimouse horseradish peroxidase. conjugate anddetected with the HRP conjugate substrate kit (Bio-Rad Laboratories).All protein bands visualized under UV light were detected after blottingto nitrocellulose.

[0070] References The following references are incorporated herein byreference as if reproduced in their entirety.

[0071] 1. Ristow, S. S., Starkey, J. R., Stanford, D. R., Davis, W. C.,and Brooks, C. G. (1985) Cell surface thiols, but not intracellularglutathione, are essential for cytolysis by a cloned murine naturalkiller cell line. Immun. Invest. 14, 401-414

[0072] 2. Hartman, B. K., and Udenfriend, S. (1969) A method forimmediate visualization of proteins in acrylamide gels and its use forpreparation of antibodies to enzymes. Anat. Biochem. 30, 391-394

[0073] 3. Andrews, A. T. (1981) Electrophoresis: theory, techniques andbiochemical and clinical applications. Oxford University Press, NewYork.

[0074] 4. Dunn, M. J. (1993) Gel Electrophoresis: Proteins. BiosScientific Publishers Ltd. Oxford, Great Britain.

[0075] 5. Steinberg, T. H., Jones, L. J., Haugland, R. P., and Singer,V. L. (1996) SYPRO orange and SYPRO red protein gel stains: One-stepfluorescent staining of denaturing gels for detection of nanogram levelsof protein. Anal. Biochem. 239, 223-237.

[0076] 6. Berggren, K., Chernokalskaya, E., Steinberg, T. H., Kemper,C., Lopez, M. F., Diwu, Z., Haugland, R. P., Patton, W. F.Background-free, high sensitivity staining of proteins in one- andtwo-dimensional sodium dodecyl sulfate-polyacrylamide gels using aluminescent ruthenium complex. Electrophoresis 21 ,2509-2521 (2000).

[0077] 7. Jones, D., T., Taylor, W. R., and J. M. Thornton. A mutationdata matrix for transmembrane proteins. FEBS letters 339, 269-275(1994).

[0078] 8. Deber, C. M., Brandl, C. J., Deber, R. B., Hsu, L. c. and X.K. Young. Amino acid composition of the membrane and aqueous domains ofintegral membrane proteins. 1986 251, 68-76 (1986).

[0079] 9. Voropei, A. V., Chernitskii, Ye. A., Konev, S. V., Krivitskii,A. P., Pinchuk, S. V., and Shchukanova, N. A. (1992)Chloroform-Dependent photoproducts of tryptophan. Biophysics 37,743-745.

[0080] 10. Pinchuk, S. V., and Vorobei A. V (1993) Spectralcharacteristics of mechanisms of forming “chloroform-dependent”tryptophan photoproducts. J. Appl. Spectrosc. 59, 711-715

[0081] 11. Edwards R. A., Jickling, G., and Turner, R. J. (2001) Thelight dependent reaction between chloroform and tryptophan. Proceedingsof the 45^(th) annual meeting of the Biophysical Society. p. 364a.

[0082] 12. Asquith, R. S., and Rivett, D. E. (1971) Studies on thephotooxidation of tryptophan. Biochim. Biophys. Acta 252, 111-116

[0083] 13. Finley, E. L., Dillon, J., Crouch, R. K., and Schey, K. L.(1998) Identification of tryptophan oxidation products in bovinealpha-crystallin. Prot. Sci. 7, 2391-2397

[0084] 14. Holt, L. A., Milligan, B., Rivett, D. E. and Stewart, F. H.H. (1977) The photodecomposition of tryptophan peptides. Biochim.Biophys. Acta 499, 131-138

[0085] 15. Sen, A. C., Ueno, N., and Chakrabarti, B. (1992) Studies onhuman lens: I. Origin and development of fluorescent pigments.Photochem. Photobiol. 55, 753-764

[0086] 16. Creed, D. (1984) The photophysics and photochemsitry of thenear-UV absorbing amino acids-I. Tryptophan and its simple derivatives.Photochem. Photobiol. 39, 537-562.

[0087] 17. Laemmli, U. K. Nature 227, 680-685 (1970)

[0088] 18. Winstone, T. L., Duncalf, K. A, and R. J. Turner.Optimization of expression and the purification by organic extraction ofthe integral membrane protein EmrE. Protein Expression and Purification26, 111-121 (2002).

[0089] 19. Oresnik, I. J., Ladner, C. L. and R. J. Turner.Identification of a twin-arginine leader-binding protein. MolecularMicrobiology 40, 323-331 (2001).

[0090] 20. Liu, M., Turner, R. J., Winstone, T. L., Saetre, A.,Dyllick-Brenzinger, M., Jickling, G., Tari, L. W., Weiner, J. H. and D.E. Taylor. Escherichia coli TehB Requires S-Adenosylmethionine as aCofactor to Mediate Tellurite Resistance. Journal of Bacteriology 182,6509-6513 (2000).

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1. A method of detecting proteins in a polyacrylamide gel, comprisingthe steps of: (a) incorporating a haloalkane into the gel, eitherpre-electrophoresis or post-electrophoresis which haloalkane reacts withtryptophan residues in the proteins to form fluorescent compounds whenilluminated with ultraviolet light; (b) electrophoretically separatingthe proteins in the gel; and (c) illuminating the gel with ultravioletlight; and (d) detecting the fluorescence of the formed compounds. 2.The method of claim 1 wherein the haloalkane is a trichloroalkane. 3.The method of claim 2 wherein the trichloroalkane is selected from thegroup consisting of chloroform, trichloroacetic acid andtrichloroethanol, or mixtures thereof.
 4. The method of claim 1 whereinthe gel is soaked in a haloalkane solution after electrophoresis.
 5. Themethod of one of claim 1 wherein the haloalkane is incorporated into thegel prior to electrophoresis.
 6. The method of claim 5 wherein the gelcomprises between about 0.02% to about 2.0% trichloroalkane (v:v) addedprior to polymerization of the gel.
 7. The method of claim 6 wherein thegel comprises between about 0.10% to about 1.0% trichloroalkane (v:v)added prior to polymerization of the gel.
 8. The method of claim 7wherein the gel comprises about 0.5% trichloroalkane (v:v) added priorto polymerization of the gel.
 9. The method of claim 4 wherein the gelis soaked in a solution comprising trichloroethanol or trichloroaceticacid, or mixtures thereof, in water or alcohols (including but notlimited to methanol) or mixtures thereof.
 10. A polyacrylamide gel forelectrophoretically separating proteins by molecular weight, the gelcomprising a haloalkane which reacts with tryptophan residues in theproteins to form compounds which fluoresce when subjected to ultravioletlight.
 11. The polyacrylamide gel of claim 10 wherein the haloalkane isselected from the group consisting of chloroform, trichloroacetic acid,and trichloroethanol, or mixtures thereof.
 12. A method of forming apolyacrylamide gel for electrophoretically separating proteins bymolecular weight, comprising the step of incorporating into the gel ahaloalkane which reacts with tryptophan residues in the proteins to formcompounds which fluoresce when subjected to ultraviolet light.
 13. Themethod of claim 12 wherein the haloalkane is selected from the groupconsisting of chloroform, trichloroacetic acid, and trichloroethanol, ormixtures thereof.
 14. The method of claim 12 wherein the haloalkane isincorporated into the gel by soaking the gel in a haloalkane solution.15. The method of claim 12 wherein the haloalkane is incorporated intothe gel prior to polymerization of the gel.